Reagents and Methods for Cancer Treatment and Prevention

ABSTRACT

The invention generally relates to the prevention and/or treatment of cancer, and, more specifically, to the treatment of tumors, including solid tumors and their metastases, without radiation or standard chemotherapeutic agents. In one embodiment, the invention involves a method comprising: a) providing a subject with tumor cells, b) removing at least a portion of said tumor cells from said subject to create removed cells, c) treating at least a portion of said removed cells ex vivo, using stimulating agents, including thapsigargin and/or thapsigargin-related compounds, so as to create treated tumor cells; and d) introducing said treated tumor cells (or fragments thereof) in vivo into the same subject to generate anticancer therapeutic effects.

This application is a divisional application of allowed U.S. applicationSer. No. 11/389,695, filed Mar. 27, 2006, which is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to the treatment of cancer, and, morespecifically, to the treatment of tumors, including solid tumors andtheir metastases, without radiation or standard chemotherapeutic agents.In a preferred embodiment, the invention relates to the prevention andtreatment of cancer through use of a cancer vaccine.

BACKGROUND

Modern cancer therapy largely involves the use of radiation, surgery andchemotherapeutic agents. However, results with these measures, whilebeneficial in some tumors, has had only marginal or no effect in manyothers. Furthermore, these approaches often have unacceptable toxicity.

Both radiation and surgery suffer from the same theoretical drawback. Ithas been recognized that, given that a single clonogenic malignant cellcan give rise to sufficient progeny to kill the host, the entirepopulation of neoplastic cells must be eradicated. See generally,Goodman and Gilman The Pharmacological Basis of Therapeutics (PergamonPress, 8th Edition) (pp. 1202-1204). This concept of “total cell kill”implies that total excision of a tumor is necessary for a surgicalapproach, and complete destruction of all cancer cells is needed in aradiation approach, if one is to achieve a cure. In practice this israrely possible; indeed, where there are metastases, it is impossible.

Moreover, traditional chemotherapeutic cancer treatments also rarelyresult in complete remission of the tumor, and the significant dosagelevels required to generate even a moderate response are oftenaccompanied by unacceptable toxicity. Anticancer agents typically havenegative hematological effects (e.g., cessation of mitosis anddisintegration of formed elements in marrow and lymphoid tissues), andimmunosuppressive action (e.g., depressed cell counts), as well as asevere impact on epithelial tissues (e.g., intestinal mucosa),reproductive tissues (e.g., impairment of spermatogenesis), and thenervous system. P. Calabresi and B. A. Chabner, In: Goodman and GilmanThe Pharmacological Basis of Therapeutics (Pergamon Press, 8th Edition)(pp. 1209-1216). The high dosage levels, and the resulting toxicity, arein large part necessitated by the lack of target specificity of theanticancer agents themselves. The drug needs to distinguish between hostcells that are cancerous and host cells that are not cancerous. The vastbulk of anticancer drugs are indiscriminate at this level, and havesignificant inherent toxicity.

Success with standard chemotherapeutics as anticancer agents has alsobeen hampered by the phenomenon of multiple drug resistance, resistanceto a wide range of structurally unrelated cytotoxic anticancercompounds. J. H. Gerlach et al., Cancer Surveys, 5:25-46 (1986). Theunderlying cause of progressive drug resistance may be due to a smallpopulation of drug-resistant cells within the tumor (e.g., mutant cells)at the time of diagnosis. J.H. Goldie and Andrew J. Coldman, CancerResearch, 44:3643-3653 (1984). Treating such a tumor with a single drugfirst results in a remission, where the tumor shrinks in size as aresult of the killing of the predominant drug-sensitive cells. With thedrug-sensitive cells gone, the remaining drug-resistant cells continueto multiply and eventually dominate the cell population of the tumor.

Treatment at the outset with a combination of drugs was proposed as asolution, given the small probability that two or more different drugresistances would arise spontaneously in the same cell. V. T. DeVita,Jr., Cancer, 51:1209-1220 (1983). However, it is now known that drugresistance is due to a membrane transport protein, “P-glycoprotein,”that can confer general drug resistance. M. M. Gottesman and I. Pastan,Trends in Pharmacological Science, 9:54-58 (1988). Phenotypically, thetumor cells show, over time, a reduced cellular accumulation of alldrugs. In short, combination chemotherapy appears not to be the answer.

Adoptive cellular immunotherapy has been proposed as an alternativetreatment methodology, using the body's own immune system in an attemptto improve target cell specificity while reducing toxicity. Theactivation and proliferation of various lymphocyte populations withlymphokines both in vivo and in vitro has been investigated, with mixeddegrees of success. For example, lymphokine-activated killer (LAK.)cells and tumor-infiltrating lymphocytes (TILs) have both been used incombination with interleukin-2 (IL-2) in the treatment of metastaticdisease. See Rosenberg et al., N. Engl. J. Med. 316:889-97 (1987);Belldegrun et al., Cancer Res 48:206-14 (1988).

Unfortunately, the inclusion of high levels of IL-2 to activate andexpand the cell populations is itself associated with significanttoxicity to the patient. Moreover, target-cell specific cell populationshave been difficult to expand in vitro, since lymphocytes cultured inhigh levels of IL-2 eventually develop an unresponsiveness to IL-2, andsubsequently exhibit a serious decline in proliferation andcytotoxicity. See Schoof et al., Cancer Res. 50: 1138-43 (1990). Thelatter problem has also impeded efforts to successfully use lymphocytesas cellular vehicles for gene therapy in man.

What is needed is a specific anticancer approach that is reliablytumoricidal to a wide variety of tumor types. Importantly, the treatmentmust be effective with minimal host toxicity.

SUMMARY OF THE INVENTION

The invention generally relates to the prevention and/or treatment ofcancer, and, more specifically, to the treatment of tumors, includingsolid tumors and their metastases, without radiation or standardchemotherapeutic agents. In one embodiment, the invention involves amethod comprising: a) providing a subject with tumor cells, b) removingat least a portion of said tumor cells from said subject to createremoved cells, c) treating at least a portion of said removed cells exvivo, using stimulating agents, including thapsigargin and/orthapsigargin-related compounds, so as to create treated tumor cells; andd) introducing said treated tumor cells in vivo into the same subject togenerate anticancer therapeutic effects. Prior to introducing saidtreated tumor cells, they may be washed (e.g. with buffer, culturemedium, etc.) to minimize the amount of the agent carried over into thesubject. In a preferred embodiment, the invention comprises a methodcomprising: a) providing a patient with tumor cells, b) removing atleast a portion of said tumor cells from said patient to create removedcells, c) exposing at least a portion of said removed cells ex vivo toan agent selected from the group consisting of thapsigargin andthapsigargin-related compounds, so as to create treated tumor cells; d)lysing said treated tumor cells to create cell fragments, and e)introducing said fragments in vivo into the same patient to generate ananticancer therapeutic effect. It is preferred that in one embodiment,prior to step (d), the treated cells are washed so as to minimize theamount of agent carried over into the next step. It is not intended thatthe present invention be limited by any particular exposure/treatmenttime for step (c). A variety of exposure times is contemplated,including exposure times between 1 second and 72 hours, more preferablybetween 30 seconds and 1 hour, still more preferably 1 minute and 30minutes. It is not intended that the presentinvention be limited by thenature of the lysing in step (d). In one embodiment, the treated tumorcells are lysed by freeze/thawing the cells. In another embodiment, alysis agent is used; lysis agents can be detergents (e.g. sodium dodecylsulfate), enzymes (e.g. an enzyme digestion buffer made up of 1 ml ofQiagen Buffer B 1, 20 mu.1 of lysozyme, 45 .mu.1 of protease and 0.35 mlof Qiagen Buffer B2), or simple solutions (e.g. phosphate buffer saline)or more complex solutions for lysing cells osmotically, e.g. inhypotonic lysis buffer (5 mM sodium phosphate pH 7.4, protease inhibitorcocktail, 5 mM DTT). In another embodiment, the cells are sonicated. Inanother embodiment, a cell extract is made (e.g. a membrane extract, acytoplasmic extract, etc.). In still other embodiments, combinations ofthese methods are used (e.g. hypotonic lysis followed by treatment witha homogenizer, (e.g. Polytron® PT 3000, Kinematica, Luzern,Switzerland). It is not intended that the present invention be limitedto how the cell fragments or cell components are introduced; they can beadministered intravenously, intramuscularly, intraperitoneally,subcutaneously, intradermally, etc. It may be preferred to administerthe fragments/extracts in certain ways for certain cancers. For example,in the case of skin cancer, it may be preferred (in one embodiment) toadminister the fragment/extracts through the skin (e.g. subcutaneously,transdermally, etc.) to treat such cancers as melanoma. In the case ofprostate cancer, it may be preferred (in one embodiment) to administerthe fragments/extracts intraperitoneally. On the other hand, in someembodiments, the fragments/extracts are administered to a site that isdistant from the tumor and into a tissue (e.g. muscle, skin, etc.) thatis unrelated to the tissue type of the tumor (e.g. breast cancer, lungcancer, etc.).

It is also not intended that the present invention be limited tointroducing only fragments/extracts in vivo; other compounds (includingbut not limited to, adjuvants, mitogens and the like) or components(including but not limited to expression constructs providing continuousor transient expression of peptides, polypeptides or proteins, orconstructs that provide continuous or transient generation of nucleicacid molecules). With regard to other compounds, the present inventioncontemplates in one embodiment, the administration of one or morecytokines before, after, or together with the fragments/extracts. Instill other embodiments, the patient may be treated before, after or atthe same time with one or more drugs that stimulate production of whiteblood cells in the bone marrow, such as the granulocyte-macrophagecolony stimulating factor (GM-CSP, generic name sargramostim, brand nameLeukine®) and granulocyte colony stimulating factor (GCSF, generic namefilgrastim, brand name Neupogen®). With regard to preferred expressionconstructs, in one embodiment, the present invention contemplatesintroducing a CD40L expression plasmid before, after, or together withthe fragment/extracts in order to create a long-lasting effect.

It is preferred, in one embodiment, that the fragments/extracts are cellfree, in order to avoid the reintroduction of live cancer cells into thepatient (however, it may be possible in some cases to reintroduce cells,rather than extracts, or extracts that contain cells, because theyrapidly die or are rapidly destroyed by the patient). In one embodiment,said tumor cells are obtained from a biopsy and the extracts areadministered to the same patient. In another embodiment, thefragments/extracts are introduced into a different patient (indeed, thefragment/extracts may be used widely on a plurality of patients). Instill a third embodiment, the cells are established as a cell line forcontinuous anticancer use; in other words, in this third embodiment,tumor cells are not taken from a patient each time they are needed, butare, instead, provided from a cell line (usually the same cancer type asthe patient's tumor). The cell line can be stored in appropriatecontainers under liquid nitrogen using conventional techniques (e.g.,DMSO, culture media, fetal calf serum, etc.). On the other hand, theymay be passed continuously in culture until use. It is not intended thatthe present invention be limited to the particular type of cancer. Avariety of cancer types are contemplated, including but not limited toskin cancer cells, prostate cancer cells, breast cancer cells, cervicalcancer cells, uterine cancer cells, pancreatic cancer cells, coloncancer cells, lung cancer cells, bone cancer cells and lymphomas (seeTable 1 for a list of illustrative cell lines). It is not intended thatthe present invention be limited to a single treatment, i.e. thefragments/extracts (with or without other compounds or components) canbe administered at intervals (e.g. once per week, twice per month, onceper month, once every six months, once per year, etc.).

It is contemplated in some embodiments where the tumor cells are removedfrom the patient, that a) standard surgery for the removal of a primarytumor (or portion thereof) is used. After b) treating the tumor cells exvivo, using stimulating agents, including thapsigargin and/orthapsigargin-related compounds, so as to create treated tumor cells; c)preparing tumor cell fragments/extracts from said treated tumor cells,and d) introducing said fragments/extracts in vivo into the samepatient, it is contemplated that an anti-metastases effect is generated.In this manner, embodiments of the present invention can be combinedwith conventional surgical procedures. For example, in one embodiment, aprimary tumor in the breast may be removed by conventional surgery (i.e.partial or complete mastectomy), and the fragments/extracts may be

TABLE 1 Designation And Origin Of Human Cell Lines And Strains¹ ORIGINCELL LINES OR STRAINS Colonic carcinoma SW1116, HCT116, SKCO-1, HT-29,KM12C, KM12SM, KM12L4, SW480 Pancreatic carcinoma BxPC-3, AsPC-1,Capan-2, MIA PaCa-2, Hs766T Colon adenoma VaCo 235 Lung carcinoma A549Prostate carcinoma PC-3, DU-145 Breast carcinoma 009P, 013T LymphomaDaudi, Raji Breast epithelium 006FA Diploid fibroblast HCS (humancorneal stroma), MRC-5 ¹The SWl 116, HT-29, SW480, Raji lympboblastoidcells, and the pancreatic lines are obtained from the American TypeCulture Collection.given to the patient post-surgery to treat metastases. In someembodiments, the treatment is done whether or not it is known that thepatient has metastases (or where metastases have been detected but thefull scope of metastatic disease is not known). In other words,protective therapy of the present invention can be applied to patientsafter conventional surgery, as well as acute therapy for knownmetastatic disease.

On the other hand, it is not always possible to remove the primarytumor. It is contemplated in some embodiments where the tumor cells areremoved from the patient, that a) standard surgery for the removal of ametastases (or portion thereof) is used. After b) treating the tumorcells ex vivo, using stimulating agents, including thapsigargin and/orthapsigargin-related compounds, so as to create treated tumor cells; c)preparing tumor cell fragments/extracts from said treated tumor cells,and d) introducing said fragments/extracts in vivo into the samepatient, it is contemplated that an anti-tumor effect against theprimary tumor (as well as residual metastatic disease) is generated. Inthis manner, embodiments of the present invention can be combined withconventional surgical procedures.

In yet another embodiment, subjects are treated in order to preventcancer. In a preferred embodiment, subjects at risk for particularcancers (whether because of age, genetic predisposition, exposure toradiation, exposure to smoke, inhalation of particulates, consumption ofmutagens, infection of HIV, etc.) are treated. In one embodiment, thepresent invention contemplates a method comprising: a) providing apatient at risk for developing cancer and a cancer cell line, b)treating cells from said cancer cell line ex vivo with an agent selectedfrom the group consisting of thapsigargin and thapsigargin-relatedcompounds, so as to create treated cancer cells; and c) introducing saidcells in vivo into said patient to generate an anticancer preventativeeffect. Of course, introducing live cancer cells may be unattractive(even though they will die or be destroyed). Therefore, in oneembodiment, the present invention contemplates a method comprising: a)providing a patient at risk for developing cancer and a cancer cellline, b) treating cells from said cancer cell line ex vivo with an agentselected from the group consisting of thapsigargin andthapsigargin-related compounds, so as to create treated cancer cells; c)preparing cell fragments/extracts from said treated cancer cells, and d)introducing said fragements/extracts in vivo into said patient togenerate an anticancer preventative effect. In this particularembodiment, the cancer cells may be from an established cell line andthe fragments/extracts can be viewed as a vaccine. It is not intendedthat the present invention be limited to a single treatment, i.e. thefragments/extracts (with or without other compounds or components) canbe administered at intervals (e.g. once per week, twice per month, onceper month, once every six months, once per year, once per five years,once per ten years, etc.) It is not intended that the present inventionbe limited to the particular type of cancer. A variety of cancer typesare contemplated, including but not limited to skin cancer cells,prostate cancer cells, breast cancer cells, cervical cancer cells,uterine cancer cells, pancreatic cancer cells, colon cancer cells, lungcancer cells, bone cancer cells and lymphomas (see Table 1 for a list ofillustrative cell lines). In a preferred embodiment, the subjects atrisk for cancer are cancer-free at the time of treatment.

The above-described vaccine may be manufactured in volume to treat apopulation. For example, in one embodiment, disease-free humans overfifty years old (or over fifty-five years old) may be administered thevaccine as a prophylactic, e.g. as a one time vaccine or repeatedly atintervals over time (e.g. every 2-5 years or 5-10 years). Indeed, thepopulation may be treated with a “cocktail” vaccine comprisingfragments/extracts of at least two different cancer types (i.e. aftertreating the cancer cells ex vivo, using stimulating agents, includingthapsigargin and/or thapsigargin-related compounds, so as to createtreated cancer cells) so as to provide a wider scope of protectionagainst cancer. The different cancer types for the “cocktail” can beselected based on increased incidence in a particular population after acertain age (e.g. increase in colon cancer and prostate cancer after agefifty-five in men; increase in breast cancer and cervical cancer afterage fifty-five in women).

In yet another embodiment, stimulating agents, including thapsigarginand/or thapsigargin-related compounds, are administered in vivo. In oneembodiment, thapsigargin and/or thapsigargin-related compounds areadministered with, in or on a carrier. In a preferred embodiment,thapsigargin is encapsulated in a delivery vehicle. It is not intendedthat the present invention be limited by the nature of the deliveryvehicle. In one embodiment, the delivery vehicle comprises a hydrophobiccarrier, e.g. hydrophobic particles, including but not limited tohydrophobic interaction beads (such beads are used as a chromatographyreagent) can deliver a powerful inflammatory signal in the presence ofthapsigargin and/or thapsigargin-related compounds. In anotherembodiment, the present invention contemplates liposomes, which are alsohydrophobic, as the delivery vehicle.

It is contemplated that the encapsulated thapsigargin (e.g.liposome-encapsulated) can be administered systemically or locally incancer patients, including but not limited to cancer patients withlocally advanced or metastatic cancers. They can be administeredintravenously, intrathecally, intraperitoneally, nasally, as well asorally. They may also be introduced by intrapulmonary administration(e.g. via inhalation or an endotracheal tube). They can also beadministered through the skin to treat cancer, including skin cancerssuch as melanoma. They can be administered alone or in combination withother compounds. They can be administered to reduce the metastatic loadin the patient prior to surgery; or they can be administered aftersurgery.

The ex vivo treatment method described above, may have advantages insome cases over direct in vivo administration (e.g. intravenousinjection). In the case of ex vivo treatment: 1) thapsigargin contactsthe appropriate target cell, namely, cancer cells; 2) exposure inculture allows for the removal of the agents prior to reintroduction ofthe fragments/extracts in the patient, i.e., the patient is exposed toonly very small amounts of thapsigargin in vivo (resulting in minimaltoxicity) and 3) lack of systemic exposure to the stimulating antigensreduces the chance of inducing antibodies to thapsigargin.

On the other hand, it is contemplated that there may be instances wheredirect in vivo administration can be used such that 1) thapsigargincontacts the appropriate target cell, namely, cancer cells; 2) thepatient is exposed to only very small amounts of thapsigargin in vivo(resulting in minimal toxicity) and 3) the chance of inducing antibodiesto thapsigargin is small. For example, where the tumor(s) is accessiblevia a procedure selected from the group consisting of endoscopy,bronchoscopy, cystoscopy, colonoscopy, laparoscopy, and catheterization,it may be possible to target cancer cells without exposing the patientto large amount of thapsigargin. Moreover, skin cancers are particularlywell-suited to direct in vivo administration of thapsigargin and/orthapsigargin-like compounds (whether in liposome formulations or otherhydrophobic formulations). Skin cancers on the surface of the skin canbe contacted with thapsigargin in situ. Skin cancers within the skin orjust below the skin can be contacted subcutaneously, intradermally, ortransdermally. In one embodiment, the present invention contemplates amethod comprising a) providing a patient having skin cancer; and b)contacting said skin cancer with thapsigargin. In one embodiment,thapsigargin is applied topically in a cream or ointment. In oneembodiment, thapsigargin is applied to the skin via a patch.

With respect to the skin, the present invention contemplates in certainembodiments exposing healthy melanocytes (or dendritic cells) tothapsigargin (e.g. via liposome delivery, a skin patch or the like) soas to trigger a protective anti-cancer immune response. While notlimited to a particular mechanism, in such an approach, it is believedthat thapsigargin introduced in the vicinity of healthy melanocytes mayactivate the immune system to inhibit melanoma progression at a distantsite (or protect against melanoma formation at a distant site).Regardless of the mechanism, it is experimentally shown (below) thattreatment with thapsigargin can be beneficial even when administered atsites distant from the cancer.

The present invention also contemplates other approaches to direct invivo administration where 1) thapsigargin contacts the appropriatetarget cell, namely, cancer cells; 2) the patient is exposed to onlyvery small amounts of thapsigargin in vivo (resulting in minimaltoxicity) and 3) the chance of inducing antibodies to thapsigargin issmall. For example, in one embodiment, the present inventioncontemplates a targeted approach to direct in vivo administrationwhere 1) thapsigargin is conjugated to a targeting molecule or moiety sothat it is brought in contact the appropriate target cell, namely,cancer cells; and thereby 2) the patient is exposed to only very smallamounts of thapsigargin in vivo (resulting in minimal toxicity andreducing the chance of inducing antibodies to thapsigargin). It is notintended that the present invention be limited to the targeting moleculeor moiety, or the nature of the conjugate. A variety of targetingmolecules and moieties are contemplated. In one embodiment, thetargeting molecule is an antibody or fragment thereof (Fab, singlechain, etc.) and conjugates are made as described by Rodwell et al.,U.S. Pat. No. 4,671,958, hereby incorporated by reference and Goers etal., U.S. Pat. No. 4,867,973, hereby incorporated by reference. It isnot intended that the present invention be limited by the nature ofantibody. Monoclonal antibodies can be used. However, humanized orcompletely human antibodies are preferred.

It is not intended that the present invention be limited by the natureof the target for the targeting molecule (i.e. nature of the antigen forthe antibody). For example, the antibody may target receptors (e.g.estrogen receptors) on human cancer cells or oncogene products on thesurface of human cancer cells. On the other hand, the present inventionis not limited to antibody targeting. In one embodiment, thapsigargincan be conjugated directly to estrogen and thereby delivered to theestrogen receptors on human cancer cells (without the use ofantibodies). Alternatively, thapsigargin can be conjugated to othersteroidal androgens (cis-androsterone, estradiol, testosterone,19-testosterone, and 5-alpha-dihydrotestosterone) or their correspondingpeptide analogs. In other embodiments, thapsigargin can be conjugated togrowth factors such as epidermal growth factor in a manner similar tothat described by Myers et al., U.S. Pat. No. 5,087,616, herebyincorporated by reference.

While not limited to any mechanism, it is believed that exposing cellsto thapsigargin and/or thapsigargin-related compounds causes the cancercells to be irnmunostimulatory. When administered to subjects havingtumors, the extracts preferably induce a tumoricidal reaction resultingin tumor regression. It should be understood that the term, “tumoricidalreaction,” as used herein, means that the tumor cells are killed, and isnot meant to be limited to any particular method by which tumor cellsare killed. For example, it may be that the tumor cells are killeddirectly (e.g., cell-cell interaction) or indirectly (e.g., release ofcytokines like interferon). With respect to cytokines, it is shownherein that the fragments/extracts described above induce cytokinesecretion in immune cells.

In one embodiment, the present invention contemplates an in vitro assayfor screening compounds for thapsigargin-like immune effects. Forexample, in one embodiment, the present invention contemplates a method,comprising a) providing cancer cells and a pre-monocytic cell line, saidpre-monocytic cell line transfected with a DNA construct encoding aNF-κB promoter driving expression of a marker protein; b) treating saidcancer cells in vitro with an agent selected from the group consistingof thapsigargin and thapsigargin-related compounds, so as to createtreated cancer cells; c) introducing said treated cancer cells to saidpre-monocytic cell line in vitro; and e) measuring the amount of saidmarker protein. In another embodiment, the present inventioncontemplates a method, comprising a) providing cancer cells and apremonocytic cell line, said pre-monocytic cell line transfected with aDNA construct encoding a NF-κB promoter driving expression of a markerprotein; b) treating said cancer cells in vitro with an agent selectedfrom the group consisting of thapsigargin and thapsigargin-relatedcompounds, so as to create treated cancer cells; c) preparing cellextracts from said treated cancer cells, d) introducing said extracts tosaid pre-monocytic cell line in vitro ; and e) measuring the amount ofsaid marker protein. It is not intended that the screening assay belimited to using only premonocytic cell lines. Monocyte-like cellslines, such as the cell line RAW, may also be used. Moreover, whenpre-monocytic cell lines are used, it is not intended that the presentinvention be limited by the particular cell line; a variety ofpre-monocytic cell lines is known including ML1, HL60, and U-937.Preferred pre-monocytic cell lines are the human pre-monocytic celllines THP-1 and MonoMac-6.

In one embodiment, the ex vivo method is further modified such that thepatient is not exposed to said fragments/extracts. Rather, immune cellsare exposed to either intact cancer cells or the fragments/extracts exvivo. For example, patient lymphocytes are removed from the patient andexposed to the treated (i.e. treated with thapsigargin and/orthapsigargin-related compounds) tumor cells or fragment/extracts ex vivoso as to generate stimulated lymphocytes; thereafter, said stimulatedlymphocytes are reintroduced to the patient with an anti-cancertherapeutic effect. It is not intended that the invention be limited bythe origin or nature of the immune cells. Preferably, they arehematopoietic cells, such as lymphocytes (e.g., tumor infiltratinglymphocytes), macrophages, dendritic cells (and the like) or cellscapable of developing into immune cells. While they may be isolated froma variety of sources, such as bone marrow (e.g., from femurs byaspiration), spleen or peripheral blood (e.g., collected with heparinand separated by Ficoll/hypaque gradient), as well as from the tumor(e.g., tumorinfiltrating lymphocytes). It is preferred that they areobtained from the lymph nodes. While they may be obtained from normal,disease-free donors, it is also preferred that they be obtained fromtumor-bearing hosts.

Definitions

As used herein, a “subject” can be a human or animal. A patient is ahuman under medical care, whether in the hospital, as an out-patient, inthe clinic, or in a doctor's office. A “subject at risk for cancer” anda “patient at risk for cancer” may be at risk for a variety of reasons(whether because of age, genetic predisposition, exposure to radiation,exposure to smoke, inhalation of particulates, consumption of mutagens,infection by HIV, etc.). For example, a person may be at risk becausehe/she is in a family with a history of cancer. On the other hand, onemay be at risk because of the results of a genetic screening assay. Withregard to the latter, detection of mutations is an increasinglyimportant area in clinical diagnosis, including but not limited to thediagnosis of cancer and/or individuals disposed to cancer (i.e. atrisk). The protein truncation test (PTT) is a technique for thedetection of nonsense and frameshift mutations which lead to thegeneration of truncated protein products. Genes associated with cancersuch as human mutL homologue and human nutS homologue (both involved incolon cancer), and BRAC1 (involved in familial breast cancer) can now bescreened for mutations in this manner, along with others. Those who arefound to have such truncation mutations (as well as other types ofmutations) are recognized to be at risk for cancer.

Thapsigargicin and thapsigargin are natural compounds (closely relatedguaianolides) that can be synthesized or (more commonly) extracted fromthe roots of Thapsia garganica L. Indeed, there are at least closelyrelated guaianolides found in Thapsia (see Table 2 below).

TABLE 2 The known range of thapsigargins found in Thapsia

Compound R¹ R² 1 Thapsigargin O-Oct But 2 Thapsigargicin O-Hex But 3Thapsitranstagin O-iVal 2-MeBut 4 Thapsivillosin A O-Ang Sen 5Thapsivillosin B O-Anq 2-MeBut 6 Thapsivillosin C O-Oct 2-MeBut 7Thapsivillosin D O-6-MeOct Sen 8 Thapsivillosin E O-6-MeOct 2-MeBut 9Thapsivillosin G O-6-MeHep 2-MeBut 10 Thapsivillosin H O-Ang or Sen Angor Sen 11 Thapsivillosin I O-Ang But 12 Thapsivillosin J O-Ival But 13Thapsivillosin K O-Sen 2-MeBut 14 Trilobolide H (S)-2-MeBut 15Nortrilobolide H But 16 Thapsivillosin F H Sen Ang, angeloyl; Sen.senecioyl; iVal, Iso-valeroyl.These 16 natural compounds fall into two series of moleculesdifferentiated by the presence of an oxygen substituent at the C-2position. In the trilobolide series of compounds, this substituent isabsent. Thapsigargin is commercially available from a number of sources:MP Biomedicals (Irvine Calif.), Sigma, Calbiochem, and Alomone Labs.Thapsigargicin is also commercially available (Calbiochem). Thapsigarginis often referred to as a sesquiterpene lactone (see structure 1, below)or sesquiterpene lactone tetraester. Thapsigargin derivatives have beenmade by a variety of strategies. For example, hydrolysis of the acetylgroup at O-10 has been performed to give deacetylthapsigargin (seestructure 2, below).

Acetylation of one of the hydroxyl groups and reduction of the lactonecarbonyl into a methylene group have also been done. As used herein,“thapsigargin-related compounds” include thapsigargin derivatives,analogues and compounds with similar activity. Thus, natural analogues(see Table 2 above) as well as synthetic analogues (see below) areintended to be within the scope of the present invention. Indeed,sesquiterpenoids generally and guaianolides specifically are intended tobe within the scope of the present invention. A number of analogues ofthapsigargin have been synthesized by alkylating or acylating O-11 andO-12 in the lactol obtained by reducing thapsigargicin. Introduction ofalpha-disposed substituents decreased the Ca(2+)-ATPase inhibitorypotency of the analogue, whereas the enzyme was more tolerant towardbeta-disposed substituents, indicating that the alpha-face of thelactone ring is in close contact with the binding site when theinhibitor is bound to the enzyme. Some analogues made synthetically havebeen shown to have potent activity (such as compound 77, below). Otheranalogues include L12ADT.

With respect to derivatives, the present invention contemplates avariety including but not limited to 8-O-debutanoylthapsigargin,8-O-(4-aminocinnamoyl)-8-0-debutanoylthapsigargin (termed ACTA). Athapsigargin CS-derivative (ZTG) was synthesized by acylatingdebutanoyl-thapsigargin with 4-azido[carboxyl-14C]benzoic acid.

With respect to compounds “having similar activity,” thapsigargin (TG)is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmicreticula. Previous enzymatic studies have concluded that Ca(2+)-ATPaseis locked in a dead-end complex upon binding TG with an affinity of <1nM and that this complex closely resembles the E(2) enzymatic state.However, compounds having similar activity need not act through the samemechanism. Drugs that might have similar effects include (but are notlimited to): 2,5-di-(tert-butyl)-1,4-benzohydroquinone, cholecystokininoctapeptide (CCK-8), cyclopiazonic acid, calcium ionophore A23187,3,3′-Diindolylmethane (DIM), ring-substituted DIMs and1,1-bis(3′-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs),N,N-Dimethyl-D-erythro-sphingosine (DMS), Econazole, inositol1,4,5-trisphosphate, and Pasteurella multocida toxin (PMT).

The present invention contemplates, as compositions, cancer cellstreated with the above compounds. In a preferred embodiment the presentinvention contemplates, as compositions, the fragments and/or extractsof these treated cancer cells.

As used herein, an “anticancer therapeutic effect” includes one or moreof the following: inhibition of cancer cell growth, increased cancercell death (a tumoricidal reaction), reduction in tumor invasiveness,reduction in overall tumor burden, reduction in local tumor burden,reduction in size of the primary tumor, prevention of metastases,reduction in the number of metastases, reduction in the size ofmetastases, and prolonged life. While it is desired that the treatmentrender the subject free of disease, it is not intended that the presentinvention be limited to curing cancer. There is therapeutic benefit evenif cancer is simply slowed. It is not intended that the presentinvention be limited to the magnitude of the effect. For example, thereduction in size of the primary tumor (or of a metastases) can be aslittle as a 10% reduction or as great as a 90% reduction (or more). Itis also not intended that the present invention be limited to theduration of the anticancer therapeutic effect. The treatment (using thevarious embodiments described herein) may result in only temporaryinhibition of cancer cell growth, temporary increased cancer cell death,temporary reduction in tumor invasiveness, temporary reduction inoverall tumor burden, temporary reduction in local tumor burden,temporary reduction in size of the primary tumor, temporary preventionof metastases, temporary reduction in the number of metastases, ortemporary reduction in the size of metastases. The temporary effects maylast weeks to months, or months to years. These parameters arerelatively easy to measure (e.g a reduction in the size of the primarytumor). With respect to prevention of metastases and prolonging life,these parameters may be measured against patient population data for theparticular tumor type, stage, and the like. As used herein, an“anticancer preventative effect” or “protective effect” comprises aneffect that reduces the incidence of new cancers. This parameter can beproved in animals and measured in humans on a population basis.

As used herein, “markers” and “labels” are detectable compounds ormoieties. They can be enzymes, fluorescent molecules, and the like.Exemplary enzymes include alkaline phosphatase, horseradish peroxidase,beta-galactosidase, glucose oxidase, a bacterial luciferase, an insectluciferase and sea pansy luciferase (Renilla koellikeri), all of whichcan create a detectable signal in the presence of suitable substratesand assay conditions.

The present invention contemplates a variety of types of “liposomes”including but not limited to cationic liposomes. It is not intended thatthe present invention be limited by the precise composition of suchliposomes. In a one embodiment, the liposomes comprise one or moreglycolipids. In a preferred embodiment, the liposomes comprise one ormore phospholipids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents exemplary data showing the effects of various compoundson the proinflammatory activity of cervical cancer HeLa cells. X Axis:control (--); thapsigargin (TG); taxol, tunicamycin (Tunic), brefeldin(BrefA), cycloheximide (CHX), and calcimycin (A23187). Y Axis: TNF-α(pg/ml).

FIG. 2 presents exemplary data showing a comparison of thapsigarginversus thaspigargicin treated HeLa cells in stimulating the release ofhuman IL-8 from the monocyte cell line THP-1. X Axis: thapsigargin orthapsigargicin concentration (μM); Y Axis: Interleukin 8 (IL-8)concentration (pg/ml).

FIG. 3 presents an illustrative list showing the prevalence of cancercell lines from various tissues known to secrete cytokines followingexposure to a thapsigargin related compound.

FIG. 4 presents exemplary data showing that thapsigargin basedimmunizations reduces tumor growth and improves survival in C57B1/6mice. Panel A: Tumor growth over time. Short Lines: Saline Controls.Open Circles: Untreated B16 cell extracts. Solid Circles: 2.5 μM B16+TGcell extracts. Panel B: Cell survival over time. Short Lines: SalineControls. Open Circles: Untreated B16 cell extracts. Solid Circles: 2.5μM B16+TG cell extracts.

FIG. 5 presents exemplary data showing that thapsigargin basedimmunizations has no effect on tumor growth in nude mice (Nu/Nu). PanelA (arrows indicate injection site): Mouse 1 and Mouse 2; B16-TG inducedinflammation. Mouse 3 and Mouse 4; B16 controls. Panel B: Cell survivalover time. Open Circles: Untreated B16 cell extracts. Solid Circles: 2.5μM B16+TG cell extracts.

FIG. 6 presents exemplary data showing reduced tumor growth after B16+TGcell extract (500 μg I.P.) immunization of C57B1/6 mice. X Axis: DaysAfter Day 0 B16-F10 melanoma cell injection. Y Axis: Tumor Area (mm²)Open Circles: Two B16+TG cell extract (Day 0 and Day 116) injection andtwo B16-F10 cell injections (Day 0 and Day 116). Closed Squares: Day 0untreated control injected with B16-F10 cells. Closed Triangles: Day 116untreated control injected with B16-F10.

FIG. 7 presents exemplary data showing reduced tumor growth after B16+TG cell extract (500 μg)/CD40L expression plasmid immunization ofC57B1/6 mice. X Axis: Days After Day 0 B16-F10 melanoma cell injection.Y Axis: Tumor Area (mm²). Open Circles: Two B16+TG cell extract/CD40Lexpression plasmid intraperitoneal injections (Day 0 and Day 116) andtwo B16-F10 melanoma cell injections (Day 0 and Day 116). Open Squares:Two B16+TG cell extract/CD40L expression plasmid intradermal injections(Day 0 and Day 116) and two B16-F10 melanoma cell injections (Day 0 andDay 116). Open Diamonds: Two B16+TG cell extract/CD40L expressionplasmid subcutaneous injections (Day 0 and Day 116) and two B16-F10melanoma cell injections (Day 0 and Day 116). Closed Squares: Day 0untreated control injected with B16-F10 melanoma cells. ClosedTriangles: Day 116 untreated control injected with B16-F10 melanomacells.

FIG. 8 presents exemplary data showing TG-treated HeLa cell extractinduction of monocyte differentiation. Panel A: Untreated monocyteshaving a rounded morphology. Panel B: Treated monocytes having anelongated and flattened morphology (Arrows).

FIG. 9 presents exemplary data showing a dose-dependent improvement ofC57B1/6 mice survival following liposomal formulations ofTG administeredon Days 7, 8, 9, 14, 15, 16, 21, 22, and 23 (arrows). Open Circles:Untreated Control after B16-F10 cell Day 0 injection (S.C.). ClosedDiamond: 0.003 mole % TG (I.D.) after B16-F10 cell Day 0 injection(S.C.). Closed Square: 0.003 mole % TG (I.D.) after B16-F10 cell Day 0injection (S.C.). Closed Triangle: 0.03 mole % TG (I.D.) after B16-F10cell Day 0 injection (S.C.).

FIG. 10 Panel A presents exemplary data showing the response of 50,000THP-1 Clone A9 to various TLR ligands as determined by a luciferasereporter system. Y Axis: relative light units (RLU) generated by thechemiluminescent properties of firefly luciferase protein.

FIG. 10 Panel B presents exemplary data showing a thapsigargin (TG) doseresponse curve using 50,000 THP-1 Clone A9 as determined by a luciferasereporter system. Y Axis: relative light units (RLU) generated by thechemiluminescent properties of firefly luciferase protein. Open Bars: TGalone. Closed Bars: 30,000 TG-treated HeLa cells. Med=Media control.

FIG. 11 presents exemplary data showing the response of MonoMac-6 Clone5 to various TLR ligands as determined by a luciferase reporter system.Y Axis: relative light units (RLU) generated by the chemiluminescentproperties of firefly luciferase protein. TG (2.5 μM). Media=Control.

FIG. 12 (SEQ ID NO:1) shows the nucleotide sequence for CD40L.

FIG. 13 (SEQ ID NOS:2 and 3) shows the CD40L cDNA prepared for ligationinto the Xbal restriction site of pVAX1.

FIG. 14 shows the map for the commercially available pVAX1 expressionplasmid.

EXPERIMENTAL

The following represents certain illustrations and embodimentscontemplated by the present invention and are not intended to belimiting.

EXAMPLE 1 Basic Laboratory Procedures

This example provides basic materials and methods that are used inExamples II-VII below. While these particular experiments utilized thespecific compounds and techniques, other similar compounds andtechniques are also capable of generating similar data.

Cell Lines

The human cervical epithelium cell line HeLa and the mouse B16-F10melanoma were purchased from American TCC and maintained in Dulbecco'smodified eagle's media [D-MEM (Cellgro®, Herndon Va.)] supplemented with10% fetal bovine serum (Sigma-Aldrich, St. Louis, Mo.). All cellstress/death-related treatments described below were performed usingHeLa cells in initial screens. The human promonocytic leukemia THP-1(ATCC, Manassas Va.) and pro-macrophage MonoMac6 cells were utilized asthe reporter antigen presenting cells utilized in this study.Ziegler-Heitbrock et al., “Establishment of a human cell line (Mono Mac6) with characteristics of mature monocytes” Int. J. Cancer 41:456-461(1988). Both lines were maintained in RPMI-1640 media (Cellgro®)supplemented with 10% fetal bovine serum. Unless otherwise noted, allcells were maintained at 37° C. in a humidified incubator supplementedwith 5% CO₂.

Compounds

The following list includes the small molecule compounds evaluated inthis study: thapsigargin, thapsigargicin (MP Biomedicals, IrvineCalif.), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic AcidTetra(acetoxymethyl) ester [BAPTA-AM, (EMD Biosciences, San Diego,Calif.)], ethyleneglycol-bis(13-aminoethyl)-N,N,N′,N′-tetraacetoxymethyl ester [EGTA-AM (EMD Biosciences)], 1,2-bis(2-aminophenoxy)ethane-N,N,N′ ,N′-tetraacetic acid (EGTA),tetrandrine, KCl, Amyloid peptide Aβ₁₋₄₂, 1-01eoyl-2-acetyl-sn-glycerol(OAG), calcimycin A23187, 2-aminoethyl diphenylborinate (2-APB),histamine, cyclopiazonic acid, 2,5-di-(t-butyl)-1,4-hydroquinone [BHQ(EMD Biosciences)], GdCl₃, nicardipine hydrochloride,1-(o-chloro-a,adiphenylbenzyl)imidazole (clotrimazole), apamin,charybdotoxin, radicicol, brefeldin A, tunicamycin, tamoxifen,ionomycin, staurosporine, cis-diammineplatinum(II) dichloride(cisplatin), paclitaxel, methotrexate, cycloheximide, and rapamycin.Unless otherwise noted, all compounds were purchased from Sigma-Aldrich.Compounds were dissolved per manufacturer's recommendations prior toaddition to HeLa cells in a range of concentrations based on thepublished literature and incubated between 1 minute to two days. Theknown mediators of inflammation pam3cys-ser-(lys)4, hydrochloride(Pam3Cys), bacterial flagellin, and lipopolysaccharide were purchasedfrom EMD Biosciences.

DNA Constructs

The cell-death inducing transgenes evaluated include human caspase 8,caspase 9, caspase 3, human insulin growth factor-1 receptorintracellular domain (IGF-1R IC), and neurokinin-1 receptor (NK1R), allcloned into the pcDNA3.1 vector (Invitrogen). The mouse CD40L (or CD154) expression plasmid was generated by creating the CD40Lcomplimentary DNA using the NCBI published nucleotide sequence of mouseCD40L (FIG. 12) and overlapping oligonucleotides encoding this sequenceflanked by XbaI restriction sites (FIG. 13) followed by polymerase chainreaction using Pfu DNA polymerase (Stratagene, San Diego Calif.). Theresulting DNA was digested with XbaI (New England Biolabs, IpswichMass.) and ligated into a similarly digested pVAX1 expression vector(Invitrogen, Carlsbad Calif.) (FIG. 14). The pVAX-CD40L construct wasverified by DNA sequencing.

Transfections

All transfections were performed using HeLa cells and Lipofectamine2000® transfection reagent (Invitrogen) per manufacturer's suggestions.NK1R-transfected cells were incubated with 1 μM substance P peptide(Sigma-Aldrich) to induce death one day following transfection.

Other Cell Stress/Death Pathways

Serum starvation was performed following incubation of HeLa cells inDMEM without serum for times ranging from 1 to 24 hours. Heat shock wasinduced by incubating cells at 42-45° C. between 1-6 hours.

Quantification of Pro-Inflammatory Cytokine Release

Following cell stress/death, resulting cells/cell debris were collected,washed 3 times with excess phosphate-buffered saline, rapidly cycledthrough freeze/thaw (−80° C. to 25° C.) three times, and quantified byUV spectrophotometry (A280). To determine the presence ofpro-inflammatory signals within stressed/dead cells, 40 μg ofstressed/dead cell were added to 50,000 THP-1 and incubated for 18hours. Supernatants were assessed for the presence of pro-inflammatorycytokines including tumor necrosis factor-α(TNF-α), interleukin 8(IL-8), and IL-12 using commercially available ELISA systems.

Generation of Thapsigargin-Dependent Inflammatory Activity

B16-F10 cells were cultured in 150 mm tissue culture plates until ˜80%confluent prior to the addition of thapsigargin (2.5 μM). Cells wereallowed to incubate for 48 hours prior to collection, three washes inexcess phosphate-buffered saline, freeze-thaw cycles, and quantificationas described above. Untreated cells were similarly harvested and bothextracts were assessed in vitro for inflammatory activity using theTHP-1 system described above prior to further use in vivo.

Mouse Immunizations and Tumor Challenge

All of the following mouse studies described were approved by the BuckInstitute Institutional Animal Care and Use Committee (IACUC) prior toinitiation. 6-8 week old female C57Bl/6 and nu/nu mice were purchasedfrom Charles River Laboratories (Wilmington Mass.). Immunizations wereperformed by injecting 500 μg of B16-F10 extract (with or withoutthapsigargin treatment) three times at two week intervals by variousroutes of immunization including subcutaneous, intraperitoneal, andintradermal routes. Live tumor challenge was performed by injecting100,000 live B16-F10 cells subcutaneously at a contralateral site twoweeks following the last booster.

EXAMPLE II Identification of A Thapsigargin-Sensitive Endogenous DangerSignal

A variety of different potential cell stress and cell death inducingagents were screened. The plant-derived thapsigargin toxin induced TNF-asecretion in a patient-derived cervical cancer cell line (HeLa) and wasthus identified for use in the various anti-cancer embodiments describedabove. In contrast, other potential cellular stressors (i.e., forexample, Taxol®, tunicamycin, brefledin A, cycloheximide, andcalcimycin) were tested but did not induce immunostimulatory activity.(See FIG. 1).

Thapsigargin was also capable of activating an antigen presenting cell(APC) in a read-out system utilizing a monocytic cell line to secretelarge amounts of the inflammatory cytokine tumor necrosis factor-α.(TNF-α). (data not shown).

EXAMPLE III Thapsigargin-Induced Secretion Of Cytokines

This example illustrates that thapsigargin treatment of cancer cellsrenders these cells immunostimulatory to antigen presenting cells(monocytes, macrophages, dendritic cells, etc.) which are activated torelease cytokines. Although it is not necessary to understand themechanism of an invention, it is believed that IL-12 plays a role in thedevelopment of cellmediated immune responses (the type of immuneresponse that may be necessary for the control of cancer and infectionby intracellular pathogens).

A thapsigargin-related compound thapsigargicin (a less hydrophobicanalog of thapsigargin) and thapsigargin activated a dose-dependentsecretion of IL-8 from the monocyte cell line THP-1. (See FIG. 2). Thesedata show that thapsigargin-related compounds are capable of activatinga potent inflammatory reaction. Thapsigargin treatment also results insecretion of IL-12, IL-8, and TNF-αin a variety of patient-derivedcancer lines. (See FIG. 3).

EXAMPLE IV In Vivo Immunization With Thapsigargin-Treated B16 Extracts

This example demonstrates that immunization with thapsigargin-treatedcells confers protection against cancer progression.

The poorly immunogenic mouse melanoma cell line B16 was treated withthapsigargin in vitro to render it pro-inflammatory (i.e, capable ofsecreting cytokines in accordance with Example III). Subsequently, micewere immunized with thapsigargin-treated B16 cells (after they werewashed, quantified, and lysed by freezing and thawing to create asolution of cell fragments) (B16+TG; 500 μg) and untreated B16 cells(B16) or saline buffer as controls, three times, once every two weeks.Two weeks following the final immunization, mice were implanted withlive B16 cancer cells and tumor progression was monitored.

B16+TG induced a transient inflammatory response in CS7B1/6 mice at thesite of immunization. This response was not seen in the saline solutionor B16 groups. The CS7B1/6 mice displayed a significantly delayed tumorprogression and an improved lifespan after immunization with B16+TG butnot after B16 or saline injections. (See FIGS. 4A and 4B, respectively).The data show that the treated animals lived, on average, almost twiceas long following tumor grafts.

A Similar experiment using nude mice (Nu/Nu) demonstrated that B16+TG(again, after the cells were washed, quantified, and lysed by freezingand thawing to create a solution of cell fragments) was ineffective inimproving survival. Thapsigargin-treated cell immunization of nude micedemonstrated a significant in situ inflammation but this was ineffectivein controlling tumor progression. (See Figure SA and Figure SB,respectively).While not necessary to the practice of the presentinvention, these data are consistent with the hypothesis that nude micepossess a genetic mutation which interferes with normal thymicdevelopment, thereby inhibiting the generation of functional Tlymphocytes.

Although it is not necessary to understand the mechanism of aninvention, it is believed that T lymphocytes, in particular the CD8+cytotoxic subset, play a role in the destruction of both neoplastic andintracellular pathogen-infected tissues. Taken together, these dataindicate that thapsigargin-treated cells activate inflammation which inturn leads to the generation of a protective T cell response in a mannersimilar to adjuvants.

EXAMPLE V Immunoprotective Memory Induced By B16+TG Injections

This example provides one embodiment wherein an immunization with B16+TG(i.e. TG-treated B 16 cells that were thereafter washed, quantified, andlysed by freezing and thawing to create a solution of cell fragments)confers long-term protection against injected melanoma.

C57B1/6 mice were immunized with B16+TG according to Example VI andsubsequently challenged with live B16-F10 melanoma cells. On Day 0 andDay 116 the B16+TG immunized mice did not develop a melanoma tumor. (SeeFIG. 6). The control mice that had not received the B16+TG extractdemonstrated typical tumor progression rates.

Alternatively, a mouse CD40L expression plasmid was co-immunized withB16+TG extract. When the immunization was performed using intradermalinjections complete tumor repression was seen at Day 0. When theimmunization was performed using either subcutaneous or intraperitonealinjections a partial tumor repression was seen at Day 0. When theanimals given a Day 0 intradermal injection were rechallenged at Day116, only a partial tumor repression was seen. (See FIG. 7)

These data demonstrate that mice immunized with TG-treated B 16 extractscan develop long lasting immunity against B 16 melanoma. In thisexperiment, the extent of this immunity, appears somewhat dependent uponthe route of administration. For example, immunizations givenintraperitoneally or subcutaneously did not completely suppress tumorgrowth following B16 melanoma cell injection at either Day 0 or Day 116.(See FIG. 7). However, the conditions have not been optimized.

In conclusion, these data show that B16+TG can protect against a livecancer cell rechallenge over at least a three month period (i.e., 116days) after the initial immunizations and challenge. These data alsosuggest that B16+TG extracts may induce the synthesis ofpro-inflammatory cytokines such as TNF-a by antigen presenting cellswhich are likely to activate a transient tumor-specific adaptive immuneresponse by B and/or T cells to delay tumor growth in vivo.

EXAMPLE VI Monocyte Differentiation Induced By B16-TG Extracts

This example presents exemplary data as to whether thapsigargin-treatedcells are capable of driving the differentiation of monocytes into othersubtypes of antigen presenting cells. Previous studies have demonstratedthat certain agents act on antigen presenting cells resulting indifferentiation into mature dendritic cells. Hertz et al., “Microbiallipopeptides stimulate dendritic cell maturation via Toll-like receptor2” J. Immunol. 166:2444-2450 (2001). Although it is not necessary tounderstand the mechanism of an invention, it is believed that dendriticcells are the main cell type responsible for determining whether toactivate an adaptive immune response or immune tolerance for any givenantigen in the body.

Undifferentiated, rounded, monocytes (See FIG. 8A) were treated with orwithout thapsigargin-treated HeLa cell extracts for 24 hours. Changes incellular morphology was then assessed (i.e., for example, inducedcellular elongation and/or flattening). The results indicate thatthapsigargin-treated cells induce a monocyte cell shape change fromprimarily rounded to flat and elongated. (See FIG. 8B, indicated byblack arrows). This shape change is believed indicative of thematuration process for undifferentiated monocytes which are generallyround in morphology into adherent cells with long cellular processestypical of macrophages or immature dendritic cells.

EXAMPLE VII Tumor Reduction Induced By A TG Liposomal Formulation

This example presents data showing that thapsigargin-containingliposomes reduce tumor progression in a dose-dependent manner.

Cationic liposomes with a total lipid content of 50 mM containingincreasing amounts of thapsigargin (0.0003, 0.003, and 0.03 mol %) wereformulated by the lipid film method followed by several cycles ofextrusion. Kuntsfeld et al., Journal of Investigative Dermatology,120:476-482 (2003). Liposomes were formulated using variousconcentrations of thapsigargin, sufficient1,2-dieoleoyl-sn-glycero-3-phosphocholine (DOPC) to bring the total to50 mol % and 50 mol % 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)in chloroform. Lipid films were generated following evaporation in arotary evaporator and re-hydrated in 5% sucrose buffer overnight.Liposomes were extruded using a 200 nM extrusion membrane and storedunder argon gas at 4° C. until use. Four groups of two female C57B1/6mice were simultaneously grafted with 1×10₅ live B16-F10 melanoma cellssubcutaneously on the left flank on day 0. Mice receivingliposome-encapsulated thapsigargin were injected intradermally with 50μl of each thapsigargin-containing liposome formulation following tumorimplantation on days 7, 8, 9, 14, 15, 16, 21, 22, and 23 at distal sitesranging from the base of the tail to the right flank and tumorprogression monitored.

Four groups of two female C57B1/6 mice were then simultaneously graftedwith 1×10⁵ live B16-F10 melanoma cells subcutaneously on the left flankon day 0. Mice receiving liposome-encapsulated thapsigargin wereinjected intradermally with 50 μl of each thapsigargin-containingliposome formulation on days 7, 8, 9, 14, 15, 16, 21, 22, and 23 atdistal sites ranging from the base of the tail to the right flank andtumor progression monitored. The data indicate thatliposome-encapsulated thapsigargin is capable of decreasing tumorprogression in a dose-dependent manner. (See FIG. 9).

EXMAPLE VIII Toxin-Induced NF-KB Promoter Systems

This example presents one embodiment of a toxin-sensitivehigh-throughput reporter (i.e. marker or label) system.

A stably transfected DNA construct encoding a NF-κB promoter drivingfirefly luciferase expression was integrated into: i) humanpre-monocytic cell line THP-1; and ii) human premonocytic cell lineMonoMac-6. Briefly, DNA encoding a concatemerized NF-κB response elementupstream of the firefly luciferase gene [Ting et al., “RIP mediatestumor necrosis factor receptor 1 activation of NF-kappaB but notFas/APO-I-initiated apoptosis” EMBO J. Nov 15;15(22):6189-96(1996)] wassubcloned into the pEAK8 vector backbone (Edge Biosystems, GaithersburgMd.) by standard molecular biology techniques. Electroporations wereperformed using 10 μg of DNA and 700,000 THP-1 or MonoMac6 cells using aBiorad Gene Pulser II electroporator (Biorad, Hercules Calif.), 0.4 cmelectrocuvettes, and 200 V, 950 μF. Following electroporation, cellswere allowed to recover for two days prior to selection with 300 ng/mlpuromycin aminonucleoside (Sigma-Aldrich) for the generation of stablytransfected clones. Luciferase expression was quantified using a TopCount NXT luminometer (Perkin Elmer, Wellesley Mass.). Briefly, 50,000reporter cells were incubated with indicated concentrations of themediators of inflammation listed for 18 hours in 96-well white platesprior to harvest and analysis.

THP-1 Clone A9

The THP-1-derived clone A9 recognition of the TLR ligands Pam-3-Cys(signaling through TLR2) and bacterial flagellin (signaling throughTLR5), but not bacterial lipopolysaccharide (signaling through TLR4) wasdetermined by monitoring NF-KB activity in 96-well white plate automatedassays during TG incubation. (See FIG. 10A).

Varying concentrations of thapsigargin were also used in the presence orabsence of pre-plated HeLa cells. These results demonstrated that CloneA9 recognized thapsigargin-treated HeLa cells at synergistically greaterlevels than either untreated HeLa cells or thapsigargin alone. (See FIG.10B).

Together, these data support the use of Clone A9 in the identificationof novel compounds and/or factors which modulate inflammation throughTLR2.

MonoMac-6 Clone C5

A second stable NF-κB reporter construct transfectant was created usingMonoMac-6 cells. In contrast to Clone A9, MonoMac-6 derived clone CSdemonstrated improved recognition of bacterial lipopolysaccharide. CloneCS also recognized Pam3Cys, flagellin, and thapsigargin-treated HeLacells. (See FIG. 11).

1.-24. (canceled)
 25. A method of quantifying immunostimulation of cancer cells, comprising: providing cancer cells and pre-monocytic cells, wherein the pre-monocytic cells are cells transfected with a DNA construct comprising a gene encoding a marker protein and an NF-κB promoter that drives expression of the marker protein; treating the cancer cells in vitro with an immunostimulant so as to create treated cancer cells; preparing cell fragments or an extract from said treated cancer cells, and contacting the pre-monocytic cells with the fragments or the extract in vitro; and measuring an amount of the marker protein formed in the expressed pre-monocytic cells.
 26. The method of claim 25 wherein the immunostimulant is an inhibitor of a Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula.
 27. The method of claim 25 wherein the immunostimulant is selected from the group consisting of thapsigargin, thapsigargicin, thapsitranstagin, a thapsivillosin, trilobolide, and nortrilobolide.
 28. The method of claim 25 wherein the immunostimulant is encapsulated in a delivery vehicle.
 29. The method of claim 25 wherein the step of preparing cell fragments is performed using at least one of freeze/thawing the cells, exposing the cells to a lysis agent, exposing the cells to a detergent, exposing the cells to an enzyme, and sonicating the cells.
 30. The method of claim 25 wherein the cancer cells are skin cancer cells, prostate cancer cells, breast cancer cells, cervical cancer cells, uterine cancer cells, pancreatic cancer cells, colon cancer cells, lung cancer cells, bone cancer cells, or lymphoma cells.
 31. The method of claim 25 wherein the cancer cells are surgically removed from a patient.
 32. The method of claim 25 wherein the cancer cells are from an established cell line.
 33. The method of claim 25 wherein the marker protein comprises firefly luciferase.
 34. The method of claim 25 wherein the pre-monocytic cell is selected from the group consisting of ML1, HL60, and U-937.
 35. The method of claim 25 wherein the pre-monocytic cell is selected from human pre-monocytic cell line THP-1 and human pre-monocytic cell line MonoMac-6.
 36. The method of claim 25 wherein the pre-monocytic cell is replaced by a monocyte-like cell.
 37. A recombinant pre-monocytic cell that is transfected with a DNA construct comprising a gene encoding a marker protein and an NF-κB promoter that drives expression of the marker protein.
 38. The recombinant pre-monocytic cell of claim 37 wherein the pre-monocytic cell is selected from the group consisting of ML1, HL60, and U-937.
 39. The recombinant pre-monocytic cell of claim 37 wherein the pre-monocytic cell is selected from human pre-monocytic cell line THP-1 and human pre-monocytic cell line MonoMac-6.
 40. The recombinant pre-monocytic cell of claim 37 wherein the pre-monocytic cell is replaced by a monocyte-like cell.
 41. A test solution comprising: a plurality of pre-monocytic cells are cells transfected with a DNA construct comprising a gene encoding a marker protein and an NF-κB promoter that drives expression of the marker protein; fragments from treated cancer cells or an extract from treated cancer cells, wherein the treated cancer cells were previously treated with an immuno stimulant; and wherein the fragments or the extract is present in the solution in an amount effective to cause expression of the marker protein via the NF-κB promoter.
 42. The test solution of claim 41 wherein the pre-monocytic cells are selected from the group consisting of ML1, HL60, U-937, THP-1, and MonoMac-6.
 43. The test solution of claim 41 wherein the cancer cells are skin cancer cells, prostate cancer cells, breast cancer cells, cervical cancer cells, uterine cancer cells, pancreatic cancer cells, colon cancer cells, lung cancer cells, bone cancer cells, or lymphoma cells.
 44. The test solution of claim 41 wherein the immunostimulant is selected from the group consisting of thapsigargin, thapsigargicin, thapsitranstagin, a thapsivillosin, trilobolide, and nortrilobolide. 